S4 Arginines Research Articles

Trapping Charge Mechanism in Hv1 Channels (CiHv1).

The majority of voltage-gated ion channels contain a defined voltage-sensing domain and a pore domain composed of highly conserved amino acid residues that confer electrical excitability via electromechanical coupling. In this sense, the voltage-gated proton channel (Hv1) is a unique protein in that voltage-sensing, proton permeation and pH-dependent modulation involve the same structural region. In fact, these processes synergistically work in concert, and it is difficult to separate them. To investigate the process of Hv1 voltage sensor trapping, we follow voltage-sensor movements directly by leveraging mutations that enable the measurement of Hv1 channel gating currents. We uncover that the process of voltage sensor displacement is due to two driving forces. The first reveals that mutations in the selectivity filter (D160) located in the S1 transmembrane interact with the voltage sensor. More hydrophobic amino acids increase the energy barrier for voltage sensor activation. On the other hand, the effect of positive charges near position 264 promotes the formation of salt bridges between the arginines of the voltage sensor domain, achieving a stable conformation over time. Our results suggest that the activation of the Hv1 voltage sensor is governed by electrostatic-hydrophobic interactions, and S4 arginines, N264 and selectivity filter (D160) are essential in the Ciona-Hv1 to understand the trapping of the voltage sensor.

The role of Phe150 in human voltage-gated proton channel

The voltage-gated proton channel Hv1 is a member of voltage-gated ion channels containing voltage-sensing domains (VSDs). The VSDs are made of four membrane-spanning segments (S1 through S4), and their function is to detect changes in membrane potential in the cells. A highly conserved phenylalanine 150 (F150) is located in the S2 segment of human voltage-gated proton channels. We previously discovered that the F150 is a binding site for the open channel blocker 2GBI. Here, we show that the Hv1 VSD voltage-dependent activation requires a hydrophobic group at position F150. We perform double-mutant cycle analysis to probe interactions between F150 and positively charged arginines in the S4 segment of the channel. Our results indicate that F150 interacts with two arginines (R2 and R3) in the S4 segment and catalyzes the transfer of the S4 arginines in the process of voltage-dependent activation.

Characterizing gating defects of SCN2A countercharge mutations in epilepsy syndromes

We are interested in the role of negatively charged residues in the S1 to S3 segments of voltage-gated sodium channels as countercharges to S4 arginine and lysine residues during gating. There are only a few studies that have investigated the functional impact of countercharge mutations associated with channelopathies of nerve or muscle. In the present work we sought to characterize the gating defects of three countercharge mutations in brain SCN2A channels that are associated with epilepsy syndromes.

Myotonia in a patient with a mutation in an S4 arginine residue associated with hypokalaemic periodic paralysis and a concomitant synonymous CLCN1 mutation

The sarcolemmal voltage gated sodium channel NaV1.4 conducts the key depolarizing current that drives the upstroke of the skeletal muscle action potential. It contains four voltage-sensing domains (VSDs) that regulate the opening of the pore domain and ensuing permeation of sodium ions. Mutations that lead to increased NaV1.4 currents are found in patients with myotonia or hyperkalaemic periodic paralysis (HyperPP). Myotonia is also caused by mutations in the CLCN1gene that result in loss-of-function of the skeletal muscle chloride channel ClC-1. Mutations affecting arginine residues in the fourth transmembrane helix (S4) of the NaV1.4 VSDs can result in a leak current through the VSD and hypokalemic periodic paralysis (HypoPP), but these have hitherto not been associated with myotonia. We report a patient with an Nav1.4 S4 arginine mutation, R222Q, presenting with severe myotonia without fulminant paralytic episodes. Other mutations affecting the same residue, R222W and R222G, have been found in patients with HypoPP. We show that R222Q channels have enhanced activation, consistent with myotonia, but also conduct a leak current. The patient carries a concomitant synonymous CLCN1 variant that likely worsens the myotonia and potentially contributes to the amelioration of muscle paralysis. Our data show phenotypic variability for different mutations affecting the same S4 arginine that have implications for clinical therapy.

Effect of Ionization State on Voltage-Sensor Structure in Resting State of the Hv1 Channel.

Voltage-gated proton-selective channels (Hv1) mediate proton extrusion during intracellular acidification. Hv1 is gated by the proton electrochemical gradient. Intracellular ionizable residues in Hv1 have been proposed to serve as proton-binding sites for pH-dependent gating, but detailed descriptions remain unclear. Here, molecular dynamics (MD) simulations were performed to investigate the effect of ionization states of charged residues on the X-ray structure of Hv1. Modification of the protonation state of acidic residues affected the resting conformation of Hv1 by disrupting salt bridges between S4 and the other segments. Upon protonation, conformational changes enabled the displacement of the S4 arginines toward the extracellular side and increased the mobility of hydrophobic residues at the gate. The aqueous crevice was considerably wider with increased hydration in the pore. Solvation free energies of the pore residues were low at the extra- and intracellular entrances, whereas the narrowest region exhibited the energy barrier for water translocation. Our MD data showed that water molecules in the upper and lower pore oriented differently. In neutral pH, the pore water oriented its dipole pointing away from the voltage-sensing domain center, whereas the opposite direction of the water dipole was observed in acidic pH.

Mechanisms Underlying the Dual Effect of Polyunsaturated Fatty Acid Analogs on Kv7.1

Polyunsaturated fatty acid (PUFA) analogs represent a new class of potential anti-arrhythmic KV7.1 and KV7.1+KCNE1 channel activators. In this study, we describe dual independent activating effects of negatively charged PUFA analogs on KV7.1 and KV7.1+KCNE1 that are dependent on discrete channel motifs. PUFA analogs are critically dependent on K326 in S6 of KV7.1 to increase the maximum conductance and critically dependent on specific S4 arginines in KV7.1 to shift the voltage dependence of channel opening toward negative voltages. Our findings provide insights into how KV7.1+KCNE1 activators may interact electrostatically both with the pore domain and the voltage-sensing domain to augment channel activity. We believe that the molecular understanding of how PUFA analogs induce dual independent activating effects is an important step toward the development of effective anti-arrhythmic drugs that target KV7.1 channels.

Replacing voltage sensor arginines with citrulline provides mechanistic insight into charge versus shape.

Voltage-dependent activation of voltage-gated cation channels results from the outward movement of arginine-bearing helices within proteinaceous voltage sensors. The voltage-sensing residues in potassium channels have been extensively characterized, but current functional approaches do not allow a distinction between the electrostatic and steric contributions of the arginine side chain. Here we use chemical misacylation and in vivo nonsense suppression to encode citrulline, a neutral and nearly isosteric analogue of arginine, into the voltage sensor of the Shaker potassium channel. We functionally characterize the engineered channels and compare them with those bearing conventional mutations at the same positions. We observe effects on both voltage sensitivity and gating kinetics, enabling dissection of the roles of residue structure versus positive charge in channel function. In some positions, substitution with citrulline causes mild effects on channel activation compared with natural mutations. In contrast, substitution of the fourth S4 arginine with citrulline causes substantial changes in the conductance-voltage relationship and the kinetics of the channel, which suggests that a positive charge is required at this position for efficient voltage sensor deactivation and channel closure. The encoding of citrulline is expected to enable enhanced precision for the study of arginine residues located in crowded transmembrane environments in other membrane proteins. In addition, the method may facilitate the study of citrullination in vivo.

When muscle Ca2+ channels carry monovalent cations through gating pores: insights into the pathophysiology of type 1 hypokalaemic periodic paralysis.

Patients suffering from type 1 hypokalaemic periodic paralysis (HypoPP1) experience attacks of muscle paralysis associated with hypokalaemia. The disease arises from missense mutations in the gene encoding the α1subunit of the dihydropyridine receptor (DHPR), a protein complex anchored in the tubular membrane of skeletal muscle fibres which controls the release of Ca2+ from sarcoplasmic reticulum and also functions as a Ca2+ channel. The vast majority of mutations consist of the replacement of one of the outer arginines in S4 segments of the α1subunit by neutral residues. Early studies have shown that muscle fibres from HypoPP1 patients are abnormally depolarized at rest in low K+ to the point of inducing muscle inexcitability. The relationship between HypoPP1 mutations and depolarization has long remained unknown. More recent investigations conducted in the closely structurally related voltage-gated Na+ and K+ channels have shown that comparable S4 arginine substitutions gave rise to elevated inward currents at negative potentials called gating pore currents. Experiments performed in muscle fibres from different models revealed such an inward resting current through HypoPP1 mutated Ca2+ channels. In mouse fibres transfected with HypoPP1 mutated channels, the elevated resting current was found to carry H+ for the R1239H arginine-to-histidine mutation in a S4 segment and Na+ for the V876E HypoPP1 mutation, which has the peculiarity of not being located in S4 segments. Muscle paralysis probably results from the presence of a gating pore current associated with hypokalaemia for both mutations, possibly aggravated by external acidosis for the R1239H mutation.

Molecular Mechanism of Polyunsaturated Fatty Acid Analogues as KV7.1-Channel Modulators

Negatively charged analogues of polyunsaturated fatty acids (PUFAs) have been shown to activate the human KV7.1+KCNE1 channel, which generates the repolarizing cardiac IKs current. Specific PUFA analogues activate both wild type KV7.1+KCNE1 and KV7.1+KCNE1 containing Long QT syndrome-associated mutations. Therefore, PUFA analogues represent a new class of KV7.1+KCNE1 channel activators that may aid in development of novel anti-arrhythmic drugs. To determine the molecular mechanism of how PUFA analogues activate KV7.1+KCNE1, we here combine electrophysiology recordings and site-directed mutagenesis with a panel of PUFA analogues with distinct properties. We identify two main effects of PUFA analogues on KV7.1 that are dependent on discrete channel motifs. The first effect is a shift in the voltage dependence of channel opening that is critically dependent on specific S4 arginines in the voltage sensor domain of KV7.1. Intriguingly, different S4 arginines are important in KV7.1 and KV7.1+KCNE1. The second effect is alteration in the maximum conductance that is critically dependent on the S6 residue K326 in the pore domain of KV7.1. Both effects appear to be primarily electrostatic. Our findings provide novel insights into how KV7.1+KCNE1 channel activators, such as PUFA analogues, simultaneously may interact with the voltage sensor domain and the pore domain to alter channel activity. Our findings also contribute pharmacological support to the hypothesis that KV7.1 and KV7.1+KCNE1 open from different conformational states.

Gating Pore Currents in Sodium Channels.

Voltage-gated sodium channels belong to the superfamily of voltage-gated cation channels. Their structure is based on domains comprising a voltage sensor domain (S1-S4 segments) and a pore domain (S5-S6 segments). Mutations in positively charged residues of the S4 segments may allow protons or cations to pass directly through the gating pore constriction of the voltage sensor domain; these anomalous currents are referred to as gating pore or omega (ω) currents. In the skeletal muscle disorder hypokalemic periodic paralysis, and in arrhythmic dilated cardiomyopathy, inherited mutations of S4 arginine residues promote omega currents that have been shown to be a contributing factor in the pathogenesis of these sodium channel disorders. Characterization of gating pore currents in these channelopathies and with artificial mutations has been possible by measuring the voltage-dependence and selectivity of these leak currents. The basis of gating pore currents and the structural basis of S4 movement through the gating pore has also been studied extensively with molecular dynamics. These simulations have provided valuable insight into the nature of S4 translocation and the physical basis for the effects of mutations that promote permeation of protons or cations through the gating pore.

Hydration and the Electric Field in the Voltage Sensing Domain of the Kv1.2 Channel: Quantum Calculations show S4 Doesn't Move, but Water and Protons Move

Quantum calculations on the VSD of Kv1.2 (3Lut pdb coordinates) show several water molecules move into the VSD when the sign of the electric field goes from positive intracellularly to negative (closed). The protein backbone remains essentially immobile; S4 does not move vertically with respect to the other transmembrane segments, but may have minimal horizontal motion (parallel to the membrane surface, were the membrane included in the calculation); side chain rearrangements, however, change some intramolecular distances. We have calculated the dipole moments of the optimized structures for several cases, as well as the structures of the water clusters (these calculations:18 water molecules, 373 atoms from the protein, from the 2nd to the 4th arginine in S4, and the complementary sections of S1, S2, and S3). Rotating two water molecules in the cluster (closed conformation) sufficed for a significant change in dipole (in most calculations, counting the dipole for the entire system; dipole changes with state as well, ≤5 D for the open configuration, approximately an order of magnitude more when closed, suggesting dipole shift is part of the sensing mechanism). Several energy minima were determined; the closed configurations were several kT lower in energy than open configurations. The water behavior resembled a phase change, with finite ΔV (volume) in its overall shift in structure with changes in electric field; there is more than one energy minimum, but the change in water density is unambiguous, although the water is not ordered in either case. Gating is coupled to water via a proton shift in the lower section of the VSD (see our other abstract).

Molecular Interactions in the Voltage Sensor Controlling Gating Properties of CaV Calcium Channels

Voltage-gated calcium channels (CaV) regulate numerous vital functions in nerve and muscle cells. To fulfill their diverse functions, the multiple members of the CaV channel family are activated over a wide range of voltages. Voltage sensing in potassium and sodium channels involves the sequential transition of positively charged amino acids across a ring of residues comprising the charge transfer center. In CaV channels, the precise molecular mechanism underlying voltage sensing remains elusive. Here we combined Rosetta structural modeling with site-directed mutagenesis to identify the molecular mechanism responsible for the specific gating properties of two CaV1.1 splice variants. Our data reveal previously unnoticed interactions of S4 arginines with an aspartate (D1196) outside the charge transfer center of the fourth voltage-sensing domain that are regulated by alternative splicing of the S3-S4 linker. These interactions facilitate the final transition into the activated state and critically determine the voltage sensitivity and current amplitude of these CaV channels.

A Point Mutation Causing Episodic Ataxia Reveals Functional Link between Voltage Sensor and Selectivity Filter in Shaker Kv Channels

Episodic ataxia (EA) constitutes itself as sporadic episodes of vertigo, migraine and ataxia and is occasionally associated with epilepsy or hemiplegic migraine. EA type 1 is caused by mutations in KCNA1. We investigated the biophysical implications of one of these mutations, F244C, in the drosophila analogue the Shaker Kv channel (numbering according to Shaker). In spite of being located in the first transmembrane helix (S1), the mutation had drastic effects on the gating currents. The gating charge-voltage relation (QV) was shifted to more depolarized potentials. Although other EA1 mutations had been linked to electromechanical coupling, this was not the case for F244, as also the conductance-voltage relations of different amino acid replacements were proportionally shifted to more depolarized potentials. Instead, we found a stabilization of the second arginine in S4 in the intermediate state of the voltage sensor dependent on the hydrophobicity of the replacement residue.One striking feature of the F244C mutant was that it led to drastic development of leak in the W434F background. This leak current passed though the main ion conducting pore, and we were able to link it to reduced C-type inactivation. We established that the C-type inactivation of the neighboring subunit was effected. F244 therefore directly links the voltage sensor movement of one subunit with the selectivity filter of its neighboring one.This work is supported by grants of the Canadian Institutes of Health Research and the Canada Research Chairs.

Molecular Determinants of Voltage Sensor Domain Activation

The voltage sensor domain (VSD) is a four transmembrane segments protein domain that confers voltage sensitivity to ion channels and other proteins. The VSD senses changes in the external potential through its highly positively charged fourth transmembrane segment (S4). The activation mechanism involves a complex, helical screw motion of S4 during which the salt bridge pattern between the S4 arginines and the negative charges of S1-S3 and of the lipid headgroups reorganizes. Together, this ratchet-like motion brings the VSD from the resting to the activated state in a series of jumps that proceed through several intermediate states. We use molecular dynamics simulations and free energy calculations to characterize the free energy landscape and the kinetic rates associated with the different steps of the activation process. To highlight the molecular determinants of this complex conformational transition, we apply techniques of machine learning and data analysis. Specifically, a custom-tailored dimensionality reduction approach is used to extract the relevant degrees of freedom describing the concerted motion of protein residues, lipids and waters.

Ion Channels and Salt Bridges: Quantum Calculations Show Unusual Effects

Calculations on voltage sensing domains (VSD) of Kv1.2 (pdb: 2A79/3Lut), with a mutation in which cysteine replaces an S4 arginine that forms a salt bridge with S2 or S3 aspartate or glutamate residues allow an alternate interpretation of the results of MTS mutation experiments, as the cavity resulting from the mutation could accommodate an MTS reagent. The position of protons in the voltage sensing domains (VSD) alters the conformation of the aromatic residues, thus affecting intracellular vs. extracellular access of MTS reagents to the cys in the mutant, rendering ambiguous the results of MTS experiments on R→C mutants. In further work on salt bridges, we have done quantum calculations (all DFT: B3LYP/6-311++G∗∗) on a salt-bridge-like “ring”, in which a carboxylic acid and guanidinium (as in arginine) are separated by two water molecules, each connecting a carboxyl oxygen and a guanidinium nitrogen. Another complete set of calculations with a third, non-ring, water, showed how a small perturbation makes a large difference. Calculations in which one water was displaced by 1A and (separate calculation) by 3A, away from its ring position, (without reoptimization) demonstrate the effect of displacing one ring water on the charges of atoms of the other water, and on several other atoms, as well as on the bond orders, suggesting the ring shows resonance. For all cases, Natural Bond Order (NBO) calculations were also done. Separate calculations performed with the acid and base neutral, and ionized, indicate conformations of the ring depend on water molecule relations to the acid/base pair, as well as their charge state. Such rings may exist in proteins, including the VSD of ion channels.

Domain III S4 in closed-state fast inactivation: Insights from a periodic paralysis mutation

Heterologous expression of sodium channel mutations in hypokalemic periodic paralysis reveals 2 variants on channel dysfunction. Charge-reducing mutations of voltage sensing S4 arginine residues alter channel gating as typically studied with expression in mammalian cells. These mutations also produce leak currents through the voltage sensor module, as typically studied with expression in Xenopus oocytes. DIIIS4 mutations at R3 in the skeletal muscle sodium channel produce gating defects and omega current consistent with the phenotype of reduced excitability. Here, we confirm DIIIS4 R3C gating defects in the oocyte expression system for fast inactivation and its recovery. We provide novel data for the effects of the cysteine mutation on voltage sensor movement, to further our understanding of sodium channel defects in hypokalemic periodic paralysis. Gating charge movement and its remobilization are selectively altered by the mutation at hyperpolarized membrane potential, as expected with reduced serum potassium.

Tracking S4 movement by gating pore currents in the bacterial sodium channel NaChBac.

Voltage-gated sodium channels mediate the initiation and propagation of action potentials in excitable cells. Transmembrane segment S4 of voltage-gated sodium channels resides in a gating pore where it senses the membrane potential and controls channel gating. Substitution of individual S4 arginine gating charges (R1-R3) with smaller amino acids allows ionic currents to flow through the mutant gating pore, and these gating pore currents are pathogenic in some skeletal muscle periodic paralysis syndromes. The voltage dependence of gating pore currents provides information about the transmembrane position of the gating charges as S4 moves in response to membrane potential. Here we studied gating pore current in mutants of the homotetrameric bacterial sodium channel NaChBac in which individual arginine gating charges were replaced by cysteine. Gating pore current was observed for each mutant channel, but with different voltage-dependent properties. Mutating the first (R1C) or second (R2C) arginine to cysteine resulted in gating pore current at hyperpolarized membrane potentials, where the channels are in resting states, but not at depolarized potentials, where the channels are activated. Conversely, the R3C gating pore is closed at hyperpolarized membrane potentials and opens with channel activation. Negative conditioning pulses revealed time-dependent deactivation of the R3C gating pore at the most hyperpolarized potentials. Our results show sequential voltage dependence of activation of gating pore current from R1 to R3 and support stepwise outward movement of the substituted cysteines through the narrow portion of the gating pore that is sealed by the arginine side chains in the wild-type channel. This pattern of voltage dependence of gating pore current is consistent with a sliding movement of the S4 helix through the gating pore. Through comparison with high-resolution models of the voltage sensor of bacterial sodium channels, these results shed light on the structural basis for pathogenic gating pore currents in periodic paralysis syndromes.

X-ray crystal structure of voltage-gated proton channel

The voltage-gated proton channel Hv1 (or VSOP) has a voltage-sensor domain (VSD) with dual roles of voltage sensing and proton permeation. Its gating is sensitive to pH and Zn(2+). Here we present a crystal structure of mouse Hv1 in the resting state at 3.45-Å resolution. The structure showed a 'closed umbrella' shape with a long helix consisting of the cytoplasmic coiled coil and the voltage-sensing helix, S4, and featured a wide inner-accessible vestibule. Two out of three arginines in S4 were located below the phenylalanine constituting the gating charge-transfer center. The extracellular region of each protomer coordinated a Zn(2+), thus suggesting that Zn(2+) stabilizes the resting state of Hv1 by competing for acidic residues that otherwise form salt bridges with voltage-sensing positive charges on S4. These findings provide a platform for understanding the general principles of voltage sensing and proton permeation.

NaV1.4 mutations cause hypokalaemic periodic paralysis by disrupting IIIS4 movement during recovery

Hypokalaemic periodic paralysis is typically associated with mutations of voltage sensor residues in calcium or sodium channels of skeletal muscle. To date, causative sodium channel mutations have been studied only for the two outermost arginine residues in S4 voltage sensor segments of domains I to III. These mutations produce depolarization of skeletal muscle fibres in response to reduced extracellular potassium, owing to an inward cation-selective gating pore current activated by hyperpolarization. Here, we describe mutations of the third arginine, R3, in the domain III voltage sensor i.e. an R1135H mutation which was found in two patients in separate families and a novel R1135C mutation identified in a third patient in another family. Muscle fibres from a patient harbouring the R1135H mutation showed increased depolarization tendency at normal and reduced extracellular potassium compatible with the diagnosis. Additionally, amplitude and rise time of action potentials were reduced compared with controls, even for holding potentials at which all NaV1.4 are fully recovered from inactivation. These findings may be because of an outward omega current activated at positive potentials. Expression of R1135H/C in mammalian cells indicates further gating defects that include significantly enhanced entry into inactivation and prolonged recovery that may additionally contribute to action potential inhibition at the physiological resting potential. After S4 immobilization in the outward position, mutant channels produce an inward omega current that most likely depolarizes the resting potential and produces the hypokalaemia-induced weakness. Gating current recordings reveal that mutations at R3 inhibit S4 deactivation before recovery, and molecular dynamics simulations suggest that this defect is caused by disrupted interactions of domain III S2 countercharges with S4 arginines R2 to R4 during repolarization of the membrane. This work reveals a novel mechanism of disrupted S4 translocation for hypokalaemic periodic paralysis mutations at arginine residues located below the gating pore constriction of the voltage sensor module.

Effects of a Novel Cav1.1-R1242G Mutation Leading to Hypokalemic Periodic Paralysis

Hypokalemic periodic paralysis (HypoPP) is an autosomal dominant muscle disorder characterized by attacks of flaccid weakness associated with a decrease in the serum potassium level. HypoPP is caused by mutations in the skeletal muscle calcium (Cav1.1)?1 or sodium (Nav1.4)? subunit. Up to date, 14 of 15 known HypoPP mutations affect the outmost two arginines in voltage-sensing S4 segments, suggesting the dysfunction of voltage sensor involved. A fresh perspective for HypoPP pathophysiology comes from the discovery of a hyperpolarization-induced inward gating pore current through the modified gating pore in Nav1.4 HypoPP mutations. The spectrum of HypoPP mutations has not been defined, so it remains to be determined whether the theory of the gating pore current could account for all cases of HypoPP. For Cav1.1 HypoPP mutations, the investigation is impeded by the difficulty of heterologous expression. Here, we describe an American family with a HypoPP type characterized by chronic progressive weakness whose affected members harbor a novel Cav1.1 R1242G mutation affecting the third arginine in S4 of domain IV. Whole-cell patch clamp recordings of the R1242G mutant showed a strong hyperpolarized shift of steady-state inactivation in GLT mouse myotube expression system. The facilitated inactivation resulting in the reduction of channel availability may account for the chronic progressive weakness of the patients. In addition, we found an outward gating pore current through the mutant voltage sensor of R1242G which might induce a reduction in the amplitude and duration of the action potential and cause the failure of muscle fiber excitability.